1. Field of the Invention
This invention relates to hapten-protein* conjugates and to fluorescent derivatives of the hapten moieties thereof. In particular, this invention relates to such conjugates and derivatives and to methods of preparing and using the same to produce novel fluorescent antibodies and to assay for certain body metabolites. The invention also relates to novel intermediates useful in the preparation of the above compounds as well as processes for the preparation of said intermediates. FNT *A small molecule, or portion of a molecule, e.g. estriol, capable of binding with a specific antibody, but incapable of generating antibodies, unless it is bound to an antigenic carrier.
2. Description of the Prior Art
An important problem in medicine is the detection and characterization of small amounts of metabolically significant substances, e.g., steroids, the catechol amines, insulin, and polypeptides, in body fluid and cells. Current methods lack sensitivity as well as specificity for the substance under assay. The results of such assays are therefore unreliable.
The importance of reliable assay methods is illustrated by the steroids, estradiol and estriol, which have a known significance in fetal abnormalities.
The fetus produces, predominantly from its adrenal gland, an androgenic molecule which serves as the precursor for estriol and is the major source for the latter in maternal serum. It is the placenta, which is a fetal product predominantly, which aromatizes the precursor molecule and hydroxylates it, resulting in estriol.
The levels of estriol in maternal blood and urine are markedly increased during pregnancy as a result of the metabolic changes. This is normal. At least one thousand to ten thousand fold increase in estriol levels occurs at term gestation, if the pregnancy is normal. However, if the fetus is in distress or is diseased, or if the placenta is immature or markedly diseased and the fetus in danger of dying, estriol levels in the maternal serum are diminished. This is true also if there is diminution of estriol in the maternal urine (assuming normal renal function) or fetal amniotic fluid.
As a result, an accurate, rapid measure of estriol levels in these body fluids would be useful in predicting fetal survival and in enabling the attending physician to judge when it may be desirable to perform a Caesarian section or to treat the mother in other fashion to save the fetus.
Estradiol is the most active of the estrogenic materials during gestation. The full impact of estradiol (and for that matter estrone) in the mother remains unknown because levels have not been accurately determinable.
For the same reason, pregnancy levels of individual estrogens from conception to 8 weeks of gestation are not known because technology does not permit accurate determination. If such determination were possible from the earliest days of conception, a standard curve could be drawn and an earlier and more reliable test for pregnancy made available.
A relatively new approach to biological assaying involves immunochemical procedures as a basis for the assay. Such procedures involve the attempted production of a synthetic antigen* which produces antibodies specific for the compound to be assayed. A known amount of such antibody and the unknown, (for example, steroid) sample, obtained from the test species, are intermixed. Theoretically, if the antibody were specific for the steroid one could then measure the amount of antibody reacting with the steroid. This amount could then be translated into the amount of test steroid present, assuming, of course, that the method of measurement was sufficiently sensitive at the concentrations involved. FNT *A molecule which generates antibodies in a host, e.g. bovine serum albumin.
Unfortunately to date no immunochemical technique has been developed which produces a reliable, accurate assay. A significant problem with previous immunochemical approaches, aside from the lack of sensitivity of available measurement techniques, has been that the synthetic antigen does not produce an antibody which is specific for the test steroid. For example, a paper published in Immunochemistry 5, 55-65 (1968) describes the synthesis of certain steroid-protein conjugates and the production of rabbit antiserum to beta-estradiol, coupled to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Antisera were tested against steroids coupled to human gamma globulin (IgG). The immunological assays employed quantitative precipitin and hapten inhibition tests.
The conjugates produced in accordance with the method described in this paper did not produce antibodies which were specific for the steroid hapten (i.e. beta-estradiol, estriol and estrone) employed in the conjugate. Antibody to estradiol-KLH brought down a non-specific precipitate with testosterone-IgG, and antibody to estriol-KLH brought down a precipitate (nonspecifically) with testosterone-IgG and with estradiol-IgG. Thus, it is impossible to achieve accurate assays employing the antibodies produced according to this method.
As will become apparent from the following description, the subject invention provides novel, purified, fluorescent antibodies which are specific for the compound under assay and novel fluorescent haptens. As a result of this achievement unique, simple, yet extremely reliable, fluorescent and radioimmune assays for a wide variety of metabolically significant substances including estrone, estradiol, estriol, the catechol amines, insulin and most polypeptides are also provided.